REGULATION OF METABOLISM IN PARASITIC HELMINTHS

寄生蠕虫代谢的调节

• 批准号: 3136886
• 负责人: Ben Gerald Harris
• 金额: $ 12.86万
• 依托单位: TEXAS COLLEGE OF OSTEOPATHIC MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1989-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3136886
• 关键词: 6 phosphofructokinase; Ascaris; actins; affinity chromatography; analytical ultracentrifugation; bioenergetics; biological information processing; cyclic AMP; gel electrophoresis; gel filtration chromatography; glycogenolysis; glycolysis; immunoprecipitation; ion exchange chromatography; ligase; malate dehydrogenase; mitochondria; pentosyltransferase; proteolysis; spectrometry

The long term objective of this project is to delineate the regulatory controls which modulate carbohydrate degradation and energy production in the muscle of the parasitic nematode, Ascaris suum. The Specific Aims are: 1) The purification, physicochemical and kinetic characterization of phosphorylase kinase from the muscle of Ascaris suum; 2) The purification, physicochemical and kinetic characterization of the phosphofructokinase kinase (PFK kinase) from the muscle of Ascaris; and 3) The utilization of the Ascaris muscle-cuticle perfusion system to study the interrelationships of the cyclic AMP-dependent and -independent regulatory systems. The basic knowledge gained from this project could provide information for the development of chemotherapeutic agents for the parasites. The enzymes will be purified by developing procedures using affinity and ion exchange chromatography, gel filtration and precipitation procedures. Criteria of purity will be by rechromatography, analytical ultracentrifugation and denaturing gel electrophoresis, which will result in determination of native molecular weight, subunit composition and molecular weight. When purified, both phosphorylase kinase and PFK kinase will be assayed by the incorporation of 32P from [Gamma-32P]ATP into substrates. Ascaris phosphorylase will be used as a substrate for phosphorylase kinase and purified phosphorylase kinase and phosphofructokinase will be used as a substrate for PFK kinase. Kinetic characterization will involve determination of apparent Km values for protein-substrates and ATP of the protein kinases. If the PFK kinase is a cyclic AMP-dependent protein kinase and it phosphorylates both phosphorylase kinase and phosphofructokinase then this will suggest that glycogenolysis and glycolysis are coordinately controlled via a cyclic AMP-mediated mechanism. In the muscle-cuticle system, perfusion of the effectors of the cyclic AMP-dependent system (serotonin) and the cyclic AMP-independent system (acetylcholine) will be conducted for various time periods followed by analysis of activation of phosphorylase, phosphorylase kinase, phosphofructokinase and PFK kinase. Correlation of these studies with those obtained on the purified enzymes should provide sufficient information to deduce the probable role of each control system in the overall metabolism of the parasite. It should also provide significant data on the possible interaction of both control systems.

本项目的长期目标是描述监管 调节碳水化合物降解和能量产生的控制， 猪蛔虫的肌肉。 具体目标 主要内容有：1）对重组蛋白的纯化、理化和动力学特性进行了研究。 猪蛔虫肌肉磷酸化酶激酶的纯化， 磷酸果糖激酶的理化和动力学特性 从蛔虫肌肉中提取PFK激酶;以及3）利用 蛔虫肌肉-表皮灌注系统，研究相互关系 环腺苷酸依赖性和非依赖性调节系统。 基本 从该项目中获得的知识可以为 开发针对寄生虫的化学治疗剂。 酶将 通过使用亲和和离子交换的开发程序进行纯化 色谱法、凝胶过滤和沉淀程序。 标准 纯度将通过再色谱法、分析超离心法和 变性凝胶电泳，这将导致确定 天然分子量、亚基组成和分子量。 当 纯化后，磷酸化酶激酶和PFK激酶都将通过 将来自[γ-32 P]ATP的32 P掺入底物中。 蛔虫 磷酸化酶将用作磷酸化酶激酶的底物， 纯化的磷酸化酶激酶和磷酸果糖激酶将用作 PFK激酶底物。 动力学表征将涉及 蛋白质底物和ATP的表观Km值的测定 蛋白激酶 如果PFK激酶是环腺苷酸依赖性蛋白 激酶，它磷酸化磷酸化酶激酶和 磷酸果糖激酶，则这将表明糖原分解和 糖酵解通过环AMP介导的 机制 在肌肉-表皮系统中， 环腺苷酸依赖系统（5-羟色胺）和环腺苷酸非依赖系统 系统（乙酰胆碱）将进行不同的时间段， 通过分析磷酸化酶，磷酸化酶激酶， 磷酸果糖激酶和PFK激酶。 这些研究与 在纯化的酶上获得的那些应当提供足够的 信息，以推断每个控制系统在系统中的可能作用。 寄生虫的整体代谢。 它还应该提供重要的 关于两个控制系统可能相互作用的数据。